Malformations involving the large arteries that exit the heart (e.g. the great vessels) are common congenital disorders. In most circumstances, the genetic basis for these abnormalities has not been identified. During embryogenesis, the great vessels arise from six pairs of bilaterally symmetric arteries embedded within the pharyngeal arches that undergo extensive remodeling to produce the complex pattern present at birth. Although the remodeling aspects have been extensively studied, the developmental origin of pharyngeal arch arteries (PAAs) and the genetic programs regulating their specification remain elusive. As severe great vessel defects are incompatible with life and milder deficiencies cause congenital cardiovascular malformations (CCMs), our long-term goal is to elucidate the cellular source of PAA endothelium and to identify genetic pathways mediating PAA establishment to potentially identify novel human disease genes. The zebrafish model organism allows for unparalleled real-time visualization and genetic dissection of PAA development. Through examination of a novel Tg(nkx2.5:ZsYellow) zebrafish reporter line, I unexpectedly discovered ZsYellow fluorescence in PAA endothelium. This observation was not anticipated, as nkx2.5 transcripts are not observed in this population. Based on these data, I postulate that PAA endothelium derives from an earlier nkx2.5+ cellular source in which ZsYellow fluorescence has persisted. Although completely unexplored, this hypothesis is supported by traditional nkx2.5 cre/loxP lineage tracing in mice. Thus, it is likely that nkx2.5 plays a conserved, yet heretofore unrecognized, role in great vessel establishment that warrants further investigation. My preliminary data also demonstrate that nkx2.5 and a requisite TGFbeta pathway component, Latent TGFbeta Binding Protein 3 (ltbp3), are required for PAA development, but dispensable for induction of the remaining vasculature. Based on compelling preliminary data, I propose to test the hypothesis that ltbp3-mediated TGFbeta signaling from the second heart field (SHF) promotes endothelial differentiation of nkx2.5-expressing PAA progenitors. Having developed new reagents for illuminating nkx2.5+ progenitors and their derivatives, I have the unique opportunity to directly test this idea. As PAA defects cause CCMs or embryonic lethality, the proposed studies are significant for informing the development of improved preventative and therapeutic approaches.